Exposure to chronic stress can activate the neuroinflammatory system in the brain, including microglial cell that may then produce the proinflammatory cytokines interleukin-1? (IL-1?), IL-6 and tumor necrosis factor-alpha (TNF?). One possible regulator of this inflammatory response is Orphanin FQ/nociceptin (OFQ/N), a neuropeptide that contributes to a variety of physiological regulatory processes, including inhibition of anxiogenic and/or depressive-like behaviors. Indeed, OFQ/N has been implicated in the regulation of systemic immunological and inflammatory processes, and in preliminary data, it was found that 14 days of chronic unpredictable stress produced a significant and augmented elevation in hippocampal IL-1?, but not TNF?, in mice lacking the precursor polypeptide for OFQ/N (ppOFQ/N). We therefore hypothesize that OFQ/N is necessary to attenuate stressor-induced activation of the brain neuroinflammatory system, including microglial activation and the synthesis and release of proinflammatory cytokines, such as IL-1?. Two Specific Aims will test this hypothesis. In Specific Aim 1, chronic stress will be imposed on mice deficient for either ppOFQ/N or the OFQ/N receptor, NOP-1. Wildtype mice (stressed and non-stressed) will be used as controls. Measures of protein and mRNA for IL-1?, IL-6, and TNF?, as well as the anti-inflammatory cytokines IL-10 and TGF? will be obtained in the prefrontal cortex, hippocampus, amygdala and hypothalamus. In addition, immunohistochemistry will determine whether there is genotype-dependent activation and cytokine localization to microglial cells, as well as stress-dependent changes in NOP-1 expression. Finally, 2-photon ex vivo hippocampal slice imaging of microglial activity will be performed on OFQ/N and NOP-1 KO and WT mice expressing eGFP in microglia to assess genotype- and/or stress-dependent effects on microglial morphology and dynamics. These biochemical and cellular observations will be complemented by behavioral analysis in Specific Aim 2. The ppOFQ/N and NOP-1 KO and WT mice will be exposed to chronic stress and then subjected to behavioral testing to assess learning and memory, as well as anxiety-like behavior. A major focus is to use a working memory version of the hippocampal-dependent T-maze spatial learning task. Excessive deficits in these behaviors in stressed KO mice, but not WT mice exposed to stress, will suggest that OFQ/N limits the neuroinflammatory response during stress in order to maintain behavioral stability. If the data lead to this interpretation, the role of IL-1? will be tested using IL-1 receptor antagonist administration in stressed and non-stressed KO and WT mice. These experiments will establish the importance of the OFQ/N ligand-receptor system as an anti-inflammatory mechanism for maintaining behavioral resilience in the face of persistent stress. Further, this will create new developments in understanding the cause and treatment of stress-related mental health disorders, and indicate that the failure of correction systems (such as OFQ/N) may result in impaired adaptation to chronic stress.